Xenotransplantation from pigs has the potential to resolve the growing shortage of human organ donors. Because of the extensive molecular incompatibilities between the donor and host, innate immunity plays a much greater role in xenograft rejection than in allograft rejection. CD47 is ubiquitously expressed and serves as a ligand of SIRP?, an inhibitory receptor on macrophages and DCs. During the current funding period, we demonstrated that the lack of cross-species interaction in CD47-SIRP? pathway largely accounts for macrophage-mediated rejection of hematopoietic and non-hematopoietic cellular xenografts. Transplantation of CD47-deficient cells induces rapid innate immune activation in syngeneic wild-type (WT) mice. Furthermore, CD47-SIRP? signal is required to repress recipient CD11hiCD8?- DC activation and induce tolerance after donor-specific transfusion (DST). Based on these and other data presented in the application, we hypothesize that the interaction between donor CD47 and host SIRP? is essential for controlling activation of SIRP?+ macrophages and DCs, and that the absence of this interaction activates host macrophages and DCs, hence stimulating anti-donor T cell responses. Here, we propose 3 specific aims to test our hypothesis. Aim 1 is to elucidate the role of CD47-SIPR? signaling in the regulation of SIRP?+ innate immune cell activation after hepatocyte xenotransplantation. We will transplant CD47 KO mouse hepatocytes into syngeneic WT mice to analyze innate immune cell activation and graft rejection induced solely by CD47 disparity. We will also transplant human CD47 transgenic vs. control pig hepatocytes into humanized mice to determine whether human CD47 expression may inhibit human innate immune responses to pig xenografts. Aim 2 is to determine the role of missing CD47-induced innate immune cell activation in T cell xenoresponses after hepatocyte transplantation. We will first address this question in allotransplant models, in which CD47 KO mice will be used to mimic CD47-incompatible xenogeneic donors. We will examine anti-donor T cell responses by in vitro and adoptive T cell transfer assays. We will then investigate the potential of human CD47 expression on pig hepatocytes to attenuate human T cell xenoimmune responses using humanized mice with a functional human immune system. Aim 3 is to determine the mechanisms by which CD47 on donor cells facilitates tolerance induction in DST plus costimulatory blockade-treated recipients. We will identify the DC and macrophage populations that are rapidly activated after CD47-deficient DST and their roles in T cell activation and tolerance induction. We will address these questions in a mouse model of CD47-deficient cardiac allotransplantation, and a pig-to-mouse xenotransplantation model that involves DST from human CD47-transgenic pigs into humanized mice. These studies are expected to provide significant insights into the mechanisms by which CD47 incompatibility activates innate and adaptive xenoimmune responses, and the potential of using human CD47 transgenic pigs as donors to facilitate xenotolerance induction and xenograft survival.